Project Summary Cancer pain therapeutics comprise more than one quarter of the total pain market. The current treatment paradigm relies on opioids, which are not effective for many patients and can have debilitating side effects for others. Despite the clear societal need for better pain medicines, there has been glacial progress in bringing new drugs to market. This shortfall is due in part to the lack of translatable model systems and tools to rapidly study relevant electrical and synaptic phenotypes associated with pain. We aim to overcome these challenges. The all-optical electrophysiology platform Optopatch, recently developed at Q-State Biosciences and comprised of engineered optogenetic proteins, custom microscopes, and software, makes it possible to simultaneously stimulate (blue light) and record (red light) electrical activity from around one hundred neurons with one millisecond temporal resolution, single cell spatial resolution and high signal-to-noise. The Optopatch platform can be used to study single-cell excitability, synaptic transmission, and network behavior with information content comparable to manual patch clamp, but at dramatically higher throughput. In this application, we propose to create an in vitro model of cancer pain via chemotherapeutics or mimicking the local tumor chemical environment experienced by sensory neurons. We will establish an Optopatch synaptic assay between primary rodent DRG neurons and dorsal horn neurons that will serve as the basis for phenotypic evaluation. Using our Optopatch excitability and synaptic assays, we will develop a ?cancer pain soup? that consists of the relevant signaling molecules. This formulation will be validated in vitro using human stem cell-derived sensory neurons as well as in vivo with nociception assays in rodents. We hypothesize that addition of this chemical mixture will lead to a phenotype of hyperexcitability or increased synaptic transmission. We will screen established tool compounds and attempt to reverse the phenotype. The Phase I application will establish the cancer pain in vitro response that can be used for phenotypic drug screening efforts in Phase II to identify novel therapeutics for cancer pain.